Angiogenesis is one of the primary factors resulting in the growth and metastasis of malignant tumors [1]. The process of angiogenesis is regulated by many factors, among which some factors promote angiogenesis, while some factors inhibit angiogenesis, and as a result, the regulation of angiogenesis is a very complicated dynamic equilibrium process [2]. Anti-angiogenesis treatment is intended to control the growth of tumor by blocking angiogenic stimulating factors or by using angiogenesis inhibitors.
At present, a large amount of angiogenic stimulating factors are known, such as, for example, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), DDR1, EphA1, EphA2, EphA8, EphB1, EphB4, EGFR, HER-2, ErbB3, MET, RON, CSF1R, KIT, PDGFR-A, PDGFR-B, TEK Tie-1, and the like, which stimulate the division and differentiation of vascular endothelial cells and the morphogenesis of blood vessels. Among these factors mentioned above, it is now known that VEGF is the most angiogenesis-specific and the most effective growth factor [3, 4].
In a hypoxic environment inside tumor tissues, VEGFs are secreted by the tumor cells, induce the division and migration of vascular endotheliocytes, and result in the establishment of a tumor vascular network. It has been demonstrated that the inhibition of VEGF may prevent angiogenesis and inhibit the growth of tumor. For this reason, VEGF and its receptors are important targets for anti-angiogenesis medicaments.
At present, anti-angiogenesis medicaments demonstrated in clinical trials to have efficacy include Bevacizumab (under the trade name of Avastin), which is able to block VEGF directly and inhibit the tumor angiogenesis. Bevacizumab was approved for marketing by FDA in 2004, and as a first-line drug for rectal cancer, it is the first marketing-approved drug that plays a role in anticarcinogenesis by inhibiting angiogenesis. Avastin is a humanized anti-VEGF monoclonal antibody, which is produced by Genentech. In a large-scale Phase III clinical trial, the combined therapy by Avastin and chemotherapy may significantly extend the survival time of the patients suffering from various types of cancers, including rectal cancer, lung cancer, breast cancer and renal cancer. [5, 6] The clinical success of Avastin show that the anti-angiogenesis treatment using tumor vascular system as the target is a clinically effective measure and can provide a new path for the tumor treatment.
Besides Avastin, several drugs for anti-VEGF signaling are also in the late phase of human clinical trial, and are expected for clinical application in the next several years. Among others, Aflibercept (also called as VEGF-Trap), developed by the cooperation between Regeneron and Sanofi-Aventis, is now under Phase III clinical trial [7]. Great progress has been achieved in the clinical treatment of tumor using anti-VEGF medicament; however, clinical trials also show that existing anti-VEGF treatment has limitations. From the point of the effect of tumor treatment, Avastin may extend the half survival time of the colon cancer patient for about 3-4 months [9, 10], and extend the half survival time of the breast cancer patient for about 7-8 months [11]; however, Avastin cannot effectively inhibit the growth of tumor blood vessel over long term. The primary causes resulting in the failure of anti-VEGF treatment or the appearance of resistance may depend on the regulation of tumor angiogenesis by a plurality of factors. Although VEGF plays an important role in angiogenesis, it is not the only angiogenesis stimulating factor. Meanwhile, owing to the heterogeneity of tumor cells, the complexity of tumor microenvironment and the compensatory response mechanism of body, when the activity of VEGF is inhibited for a long period of time, other angiogenesis stimulating factors would be expressed [12], and thus the growth of tumor blood vessel is no longer dependent on VEGF signaling path.
The variation of angiogenesis factors expressed by the tumor was studied during anti-VEGFR2 treatment for pancreatic tumor by Hanahan's group, indicating that the expression of several genes changed during anti-VEGF treatment, in which the expression of FGF-2 significantly increased. It has been shown that the expression of FGF, especially FGF-2, increased significantly in the tumor resistant to anti-VEGF treatment so that angiogenesis was activated again and the tumor repopulation was inhibited after blocking FGF signal pathway [13]. It may be seen that the over-expression of FGF-2 is closely related to the ability of tumor to escape from anti-VEGF treatment.
At present, some progress has been made in the aspect of dual- or multi-target antagonism using small molecule medicament, demonstrating that the anti-tumor effect by simultaneous antagonism of VEGF and FGF-2 is better than the single target anti-tumor therapy [14]. However, unexpected side effect may be resulted by small molecule multi-target antagonists due to the lack of specificity, and sometimes, such side effect will be shown only in the late phase of clinical trial, and thus, it is of great risk. While, macromolecule protein medicaments, especially Fc fusion protein and monoclonal antibody, have advantages, which are not possessed by the small molecule medicaments, such as high specificity and long in vivo half-life etc., which make them become the hot area for the research and development of medicament.
Fibroblast growth factor (FGF) is a heparin-binding growth factor family, which has 22 family members in the mammals (FGF 1-14, 16-23). FGF has many important biological functions, such as cell multiplication, differentiation, migration, angiogenesis and tumorigenesis. FGF exerts many biological functions by binding and activating the cell surface FGF receptor (FGFR). (See, for example, Eswarakumar et al. Cytokine Growth Factor Rev. 16: 139-149, 2005). Fibroblast growth factor receptor (FGFR) is the receptor that binds the family members of fibroblast growth factor. A part of fibroblast growth factor receptor is involved in the disease process. In the mammals, there are four FGFR genes: fgfR1-fgfR4. The fibroblast growth factor receptor is composed of extracellular domain, transmembrane domain and intracellular domain.
The family members of FGFR are different from each other in the term of ligand binding properties and kinase domains. However, the extracellular domains thereof are similar. There are three immunoglobulin-like (Ig-like) domains contained in their extracellular domains: the first Ig-like domain, the second Ig-like domain and the third Ig-like domain, and there is also a sequence contained between the first and the second Ig-like domain. Said sequence contained between the first and the second Ig-like domain is referred herein as the intermediate functional sequence region of the Ig-like domain of FGFR. Said intermediate functional sequence region comprises a region of acidic amino acids, referred as acidic box (AB).
No macromolecule fusion protein has been reported so far to be successfully constructed to block both VEGF and FGF. Although a plurality of angiogenesis-inhibitory fusion proteins have already been reported, for example, FGFR-based fusion protein (WO/2008/065543), Notch3-based fusion protein (WO/2010/021729), VEGFR-based fusion protein (WO/2010/105573), LK8-based fusion protein (WO/2008/075833) etc., all these fusion proteins are directed to a single target, and angiogenesis inhibition is realized by the fusion of a part of a single angiogenesis inhibitor and immunoglobulin Fc segment. In the prior art, no fusion protein has been reported to achieve an angiogenesis inhibition effect by inhibiting dual targets through successful fusion of two inhibitory units of blood vessel.